Mesenchymal precursor cell

ABSTRACT

A method of enriching mesenchymal precursor sells including the step of enriching for cells based on at least two markers. The markers may be either i) the presence of markers specific for mesenchymal precursor cells, ii) the absence of markers specific for differentiated mesenchymal cells, or iii) expression levels of markers specific for mesenchymal precursor cells. The method may include a first solid phase sorting step utilizing MACS recognizing expression of the antigen to the STRO-1 Mab, followed by a second sorting step utilizing two color FACS to screen for the presence of high level STRO-1 antigen expression as well as the expression of VCAM-1.

This invention relates to the enrichment of mesenchymal precursor cellsusing a combination of cell surface markers, and to a cell population ofmesenchymal precursor cells.

Mesenchymal cells are derived from a number of tissues and act as thesupportive structure for other cell types. Bone marrow for instance ismade of both haematopoietic and mesenchymal derived cells. Themesenchymal cells include endothelial cells that form the sinuses andadvetitial reticular cells that have characteristics consistent withadipocytes, fibroblasts and muscle cells.

It is believed that certain mesenchymal precursor cells (MPCs) areresponsible for the formation of mesenchymal cells. In the bone MPCs arethe formative pluripotent blast cells that are believed to be capable ofdifferentiating into any of the specific types of connective tissues(ie. the tissue of the body that support the specialised elements,particularly adipose, areolar, osseous, cartilaginous, elastic andfibrous connective tissues) depending upon the various environmentalinfluences.

Purification or at least enrichment of MPCs is desirable for a varietyof therapeutic reasons. The reasons include regeneration of missing ordamaged skeletal tissue, enhancing the implantation of various plasticor metal prosthetic devices through the attachment of the isolated andculturally expanded marrow derived mesenchymal cells onto the poroussurfaces of the prosthetic devices, which upon activation and subsequentdifferentiation of marrow-derived mesenchymal cells produce naturalosseous bridges.

Composite grafts of cultured mesenchymal cells might be used to augmentthe rate of haematopoietic cell reserve during bone marrowtransplantation.

A class of defects that may be repaired by cultured marrow-derivedmesenchymal cells expanded from the MPCs of the present invention is theclass of large skeletal defects in bone caused by injury or produced bythe removal of large sections of bone infected with tumour. Under normalcircumstances this type of defect does not heal and creates nonunion ofthe bone. This type of defect may be treated by implanting culturedmesenchymal cells contained in calcium phosphate ceramic vehicles intothe defect site.

A second class of defect that may be repaired by cultured marrow-derivedmesenchymal cells expanded from the MPCs of the present invention, isthe damaged articular cartilage generated by trauma or by diseases suchas osteoarthritis and rheumatoid arthritis. Under normal circumstances,damage to articular cartilage does not heal except in very youngindividuals where the underlying bone is also damaged so that a bloodywound is created. It is projected by the present invention that thistype of defect can be treated by implanting cultured marrow derivedmesenchymal cells into the defect. The cells will be formatted incarriers which will hold the cells in the defect and present them in amanner (round cell morphology) that they differentiate intochondrocytes.

It is not clearly understood why composite grafts of culturedmesenchymal cells and ceramic induce recruitment of haematopoietic stemcells and other marrow elements, however, the fact that this does occurallows for the use of these grafts in a way to sequester haemopoieticstem cells and generate a haematopoietic stem cell reservoir. Thereservoir of haematopoietic stem cells can then be used in clinicalapplications such as marrow transplantation as an alternative method forharvesting haematopoietic stem cells.

Another potential use for purified cells is as a means of gene therapy,by the introduction of exogenous nucleic acids for the expression oftherapeutic substances in the bone marrow—see U.S. Pat. No. 5,591,625 byGerson et al.

A purified source of MPCs is desirable for a number of reasons. Onemajor reason is that if there is a mixed population, MPCs will respondto signals elicited by other cells to behave in a manner that might notbe desired. Thus, for example, a contaminating cell might express acytokine that evokes differentiation into adipose tissue, whereas onemay require the cells for bone formation, in which case the usefulnessof the MPCs is somewhat limited. Additionally for a reason similar tothat given above, purified progenitor cells tend to be easier to handleand manage than less purified cells.

There have been many attempts at purifying or significantly enrichingMPCs, however significant enrichment has until the present invention notbeen achieved. In contrast to the haemopoietic system, in which stemcells can be physically separated based upon differences in theirexpression of cell surface markers, the cell surface antigenic phenotypeof MPCs remains relatively poorly defined. A further problem ofpurification of MPCs is a result of the physical association betweenmesenchymal cells and other cell types.

The bone and bone marrow (BM) tissues contain a phenotypically diversepopulation of stromal cell lineages that are currently thought to arisefrom a rare and primitive population of multi-potential mesenchymalprecursor cells (MPC) [Owen, 1985; Owen and Friedenstein, 1988]. Bonemarrow MPC can be readily measured by their ability to form adherentclonogenic clusters composed of fibroblastic-like cells (CFU-F:colony-forming-unit-fibroblast) in short-term liquid culture[Friedenstein et al, 1970; Castro-Malaspina et al, 1980]. In vitrostudies have documented variations in the morphology and proliferativecapacity of different BM MPC clones [Friedenstein et al, 1970; 1976;Castro-Malaspina et al, 1980; Owen et al, 1987; Bennett et al, 1991;Simmons and Gronthos, 1991]. The heterogeneous nature of the BM MPCpopulation was further demonstrated in studies where culture expandedMPC clones displayed different developmental potentials in the presenceof glucocorticoids or when transferred into ectopic sites in vivo[Friedenstein et al, 1980; Owen et al, 1987; Bennett et al, 1991].Collectively, these studies support the concept of a stromal cellhierarchy of cellular differentiation by analogy with the haemopoieticsystem.

Given the extensive literature regarding the characterisation ofhaemopoietic stem cells and their progeny there has been little progresstowards the identification of the various elements which constitute thebone marrow stromal precursor compartment. This is due in part to thelow incidence of MPC in aspirates of marrow (0.05% to 0.001%)[Castro-Malaspina et al 1980; Simmons and Torok-Storb, 1991a; 1991b;Falla et al, 1993; Waller et al, 1995a], and because of the paucity ofantibody reagents that allow for the precise identification andisolation of the MPC population. Stromal precursor cells have beenpartially enriched from bone marrow aspirates through their binding todifferent lectins such as soya bean agglutinin and wheat germ agglutininor by using a negative immunoselection process based on their lack ofexpression of various cell surface antigens restricted to the myeloid,erythroid and lymphoid cell lineages [Simmons and Torok-Storb 1991a;1991b; Simmons et al, 1994; Rickard et al, 1996]. However, theinefficiency of these selection strategies has resulted in the presenceof contaminating populations of accessory cells and haemopoieticprogenitor cells. Moreover, a major difficulty in using techniques suchas fluorescense activated cell sorting (FACS) to positively select forpure populations of MPC is that they share many common antigens with HSCincluding early developmental markers such as the human CD34 antigen andthe murine stem cell antigen-1.

Recent advances in the study of human stromal stem cell biology havebeen attributed to the development of novel monoclonal antibodies (Mabs)which recognise antigens on BM MPC that are correspondingly not reactivewith haemopoietic progenitors. We have previously described a monoclonalantibody, STRO-1 which identifies an as yet unidentified 60 kDa cellsurface antigen expressed on all assayable MPC in aspirates of adulthuman BM [Simmons and Torok-Storb, 1991a]. The majority of the STRO-1⁺bone marrow mononuclear cells (BMMNC) (approximately 90%) have beenidentified as late stage glycophorin A⁺ erythroblasts. The MPCpopulation are restricted to the minor population of STRO-1⁺ cells whichlack glycophorin A [Simmons and Torok-Strob, 1991a]. Importantly, STRO-1demonstrates no detectable binding to haemopoietic progenitors (CFU-GM,BFU-E, BFU-Meg, CFU-GEMM) nor to their precursors (pre-CFU) [Simmons andTorok-Storb, 1991a; Gronthos and Simmons, unpublished observations].

A systematic examination of the immunophenotype of MPC derived fromadult human BM has previously been performed using two-color FACSanalysis [Simmons et al, 1994]. A number of antigens were shown to becoexpressed with STRO-1 by essentially all BM MPC. These included theendopeptidases CD10 and CD13 and the adhesion molecules Thy-1 (CDw90),VCAM-1 (CD106) and various members of the β1 (CD29) integrin family[Simmons et al, 1994]. This is in accord with the data of Terstappen andcolleagues regarding the antigenic phenotype of human foetal BM MPC[Waller et al, 1995].

SUMMARY OF THE INVENTION

This invention arises from the finding that enrichment of mesenchymalprecursor cells is greatly enhanced by the use of two markers specificfor mesenchymal cells, that can be used to recognise early cells. Tothis end it will be appreciated that MPCs are early cells that aresubstantially at a pre-expansion stage of development and hence areprecursors to mesenchymal stem cells in which a significant number ofthe population have expanded and are therefore incapable of furtherexpansion. Thus, MPCs are cells that have yet to differentiate to fullycommitted mesenchymal cells. These cells need not however be stem cellsin a strict sense, in that they are necessarily able to differentiateinto all types of mesenchymal cells. There is a benefit in having anenriched pool of MPCs that are able to differentiate into bone formingcells only, in that these precursor cells have a greater proliferationpotential. In particular in accordance with the present inventionbecause the proportions of MPCs in the harvested population is large,the extent to which the population can be expanded is greatly enhanced.

The present invention provides an enrichment several orders of magnitudebetter than the best method known to the inventors before the presentinvention. The inventors have shown that an enriched population in whichup to 50% of the MPCs can form colonies of ten or more cells can beachieved using the present invention. In contrast, the citationsindicate that the best method known up until now has only achieved anenrichment of up to 0.01% cells capable of forming colonies. It is to benoted that as discussed herein the presence of MPCs is based upon theircolonigenic capacity, as determined by the presence of colonies of tenor more cells in liquid culture seeded with single cells after havingbeen grown for 14 days.

In a broad form of a first aspect the invention could be said to residein a method of enriching mesenchymal precursor cells (MPCs) the methodincluding the steps of enriching for cells based on at least twomarkers, said markers being either the presence of, or expression levelsof markers specific for mesenchymal precursor cells on the one hand, orabsence of marker or levels of expression specific for differentiatedmesenchymal cells on the other hand.

The preferred source of material for enrichment is bone marrow, and thusin a one form the method is limited to the enrichment of bone marrowderived mesenchymal stem cells. It is also likely that the method ofthis first aspect of the invention might be used to enrich stromal stemcells from other sources such as blood, epidermis and hair follicles. Itis proposed that mesenchymal precursor cells isolated from, for example,skin should have the same potential as those cells isolated from bonemarrow. An advantage in isolating cells from skin is that the harvestingis far less invasive than the harvesting of a sample of bone marrow.

It is anticipated that a proportion of the population purified will bestem cells, however, it is not yet known how to separate these stemcells from the MPC population. It has been observed however that asubpopulation has a much greater capacity to divide than others, andperhaps this subpopulation represents the stem cells. It is estimatedthat approximately 10 to 20% of the MPCs isolated by the illustratedmethod of this invention are stem cells.

It is preferred that a significant proportion of the MPCs are capable ofdifferentiation into at least two committed cell types selected from thegroup including but not limited to adipose, areolar, osseous,cartilaginous, elastic and fibrous connective.

It has been found that it is possible to purify MPCs by the above methodto a degree where these cells are present in a purified population ofwhich 50% of the MPCs can form colonies of ten or more cells. Thereforethe method may result in a cell population in which at least 1% of thecells are MPCs that are colony forming, preferably at least 5% of thecells are MPCs that are colony forming, more preferably at least 10% ofthe cells are MPCs that are colony forming, and most preferably at least40% of the cells are MPCs that are colony forming.

The nearest known purification is that by Pittenger et al. (Science 284;143–147) where cells had been enriched using a Percoll gradient. Theseworkers were only able to get colony forming units from 0.001–0.01% ofcells. The present technique therefore results in a very significantenrichment when compared to these attempts.

The present invention is also to be contrasted to the enrichedpopulations described by Caplan et al. in U.S. Pat. No. 5,837,539 whodescribes a method for the isolation, purification and culture expansionof mesenchymal stem cells which is said to give compositions havinggreater than 95% human mesenchymal stem cells. It is to be noted thatthe figure of 95% relates to populations of expanded mesenchymal stemcells, and is likely to reflect a lower number of colony forming unitsbecause the cells are at least partially expanded. Thus, Caplan startswith a population of BM cells comprising about 1 in 1000 MPCs andexpands the population and then purifies the at least partially expandedpopulation. In contrast the present invention can result in a populationof about 1 in 2 cells that are able to form colonies of at least 10MSCs.

Preferably the method includes enriching by selecting for the positiveexpression of at least one marker and more preferably both markers areselected for positive expression. These markers are most convenientlycell surface markers. The markers might be selected from a group ofsurface markers specific for MPC including but not limited to LFA-3,THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, CD49b/CD29,CD49c/CD29, CD49d/CD29, CD29, CD18, CD61, 6-19, thrombomodulin, CD10,CD13, SCF, and the antigen recognised by STRO-1.

The marker might be absence of various surface markers indicative ofcommitment, such as CBFA-1, collagen type II, PPARγ2, glycophorin A.

In one preferred form at least one of the markers is the antigenrecognised by STRO-1, and in particular the high level of expression ofthat antigen.

In another preferred form at least one of the markers is VCAM-1.

In one very specific form the two markers are the antigen recognised bySTRO-1 and VCAM-1.

The specificity of the markers used in this process is not absolute.Thus even the most preferred markers occur on cell types other thanmesenchymal cells, however their expression on the cell surfaces ofother cell types is limited.

It will be understood that recognition of cells carrying the cellsurface markers that form the basis of the separation can be effected bya number of different methods, however, all of these methods rely uponbinding a binding agent to the marker concerned followed by a separationof those that exhibit binding, being either high level binding, or lowlevel binding or no binding. The most convenient binding agents areantibodies or antibody based molecules, preferably being monoclonalantibodies or based on monoclonal antibodies because of the specificityof these latter agents. Antibodies can be used for both steps, howeverother agents might also be used, thus ligands for these markers may alsobe employed to enrich for cells carrying them, or lacking them.

The antibodies may be attached to a solid support to allow for a crudeseparation. The separation techniques should maximise the retention ofviability of the fraction to be collected. Various techniques ofdifferent efficacy may be employed to obtain relatively crudeseparations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS.

The method might include the step of making a first partially enrichedpool of cells by enriching for the expression of a first of the markers,and then the step of enriching for expression of the second of themarkers from the partially enriched pool of cells.

It is preferred that the method comprises a first step being a solidphase sorting step, based on recognition of one or more of the markers.The solid phase sorting step of the illustrated embodiment utilises MACSrecognising high level expression of STRO-1. This then gives an enrichedpool with greater numbers of cells than if a high accuracy sort was usedas a first step. If for example FACS is used first, many of the MPCs arerejected because of their association with other cells. A second sortingstep can then follow using an accurate separation method. This secondsorting step might involve the use of two or more markers. Thus in theillustrated embodiment two colour FACS is used to recognise high levelexpression of the antigen recognised by STRO-1 as wells as theexpression of VCAM-1. The windows used for sorting in the second stepcan be more advantageously adjusted because the starting population isalready partially enriched.

It will be understood that the invention is not limited to theenrichment of cells by their expression of only two markers and it maybe preferred to enrich based on the expression of three or more markers.

The method might also include the harvesting of a source of the stemcells before the first enrichment step, which in the most preferredsource comprises the step of harvesting bone marrow cells, using knowntechniques.

The preferred source of such cells is human, however, it is expectedthat the invention is also applicable to animals, and these mightinclude domestic animals or animals that might be used for sport.

In a broad form of a second aspect the invention could be said to residein an enriched population of mesenchymal precursor cells as purified bya method according to the first aspect of the invention.

It has been found that it is possible to purify MPCs to a degree wherethe purified population contains 50% of these cells that are capable offorming colonies of 10 or more cells.

In a broad form of a third aspect the invention could also be said toreside in a cell population in which at least 1% of the cells are MPCsthat are colony forming, preferably at least 5% of the cells are MPCsthat are colony forming, more preferably at least 10% of the cells areMPCs that are colony forming, and most preferably at least 40% of thecells are MPCs that are colony forming.

The cells of the enriched population preferably carry at least twomarkers selected from a group of surface markers specific formesenchymal precursor cells including LFA-3, THY-1, antigen identifiedby STRO-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, CD49b/CD29,CD49c/CD29, CD49d/CD29, CD29, CD18, CD61, 6-19, thrombomodulin, CD10,CD13 and SCF. Most preferably the cells carry the antigen identified bySTRO-1 and VCAM-1.

It will also be understood that in a fourth aspect the inventionencompasses a composition including the purified MPCs or a compositionmade from the purified MPCs.

The purified population of the second or third aspects of the invention,or the composition of the fourth aspect of the invention might be usedin the formation and repair of bones, and as such a combination of MPCsas well as a suitable support may be introduced into a site requiringbone formation. Thus, for example, skeletal defects caused by boneinjury or the removal of sections of bone infected with tumour may berepaired by implanting cultured MSCs contained in calcium phosphateceramic vehicles into the defect site. For appropriate methods andtechniques see Caplan et al. in U.S. Pat. No. 5,226,914 and U.S. Pat.No. 5,837,539, both of which use cruder preparations of stem cells.

In addition, the enriched population or composition may be used toassist in anchoring prosthetic devices. Thus, the surface of aprosthetic device such as those used in hip, knee and shoulderreplacement, may be coated with the enriched MPCs prior to implantation.The MPCs may then differentiate into osteogenic cells to thereby speedup the process of bony ingrowth and incorporation of the prostheticdevice (see Caplan et al. in U.S. Pat. No. 5,226,914 and U.S. Pat. No.5,837,539).

The enriched population or composition might also be used in genetherapy so that, for example, an enriched population may have exogenousnucleic acid transformed into it and then such a population may beintroduced into the body of the patient to treat a disease or condition.Alternatively it might be used for the release of therapeutics. Forappropriate techniques we refer to U.S. Pat. No. 5,591,625 by Gerson etal. which uses cruder preparations of stem cells.

Alternatively the enriched population or composition may be used toaugment bone marrow transplantation, wherein the composition containingpurified MSCs can be injected into a patient undergoing marrowtransplantation prior to the introduction of the whole marrow. In thisway the rate of haemopoiesis may be increased, particularly followingradiation or chemotherapy. The composition might also encompass amixture of MPCs and haemopoietic cells which may be useful inradiotherapy or chemotherapy.

FIGURE LEGENDS

FIG. 1 The frequency histogram represents the immunofluorescenceanalysis by FACS of BMMNC isolated by MACS on the basis of STRO-1 (FITC)expression: STRO-1^(dull) cell fraction (A); STRO-1^(intermediate) cellfraction (B); STRO-1^(bright) cell fraction (C); The histogram is basedon 10⁴ events collected as list mode data.

FIG. 2 Dual-colour flow cytometric analysis of VCAM-1 (PE) expression bySTRO-1⁺ (FITC) BMMNC isolated by MACS. The dot plot histogram represents5×10⁴ events collected as listmode data. STRO-1^(bright)/VCAM-1⁺ cellswere sorted by FACS (rectangle), which represented approximately 0.1% ofthe total BMMNC population (A). The incidence of clonogenic cells (B)colonies (>50 cells) and (C) colonies+clusters (>10<50 cells) based onSTRO-1^(bright)/VCAM-1⁺ expression. The frequency of clonogenic cellswas determined by limiting dilution analysis (24 replicates per cellconcentration) employing Poisson distribution analysis.

FIG. 3 Characterization of BM MPC. (A) Light microscopic examination ofthe freshly sorted cells revealed a homogenous population of large cellswith heterochromatic nuclei and prominent mucleoli, a granular cytoplasmand numerous blel-like projetions of the cell membrane (magnified 40×).(B) Transmission electron micrograph of STRO-1^(bright)/VCAM-1⁺ sortedcells isolated directly from BM (magnified 1000×). (C)Immunohistological staining of cytospin preparations of the sortedSTRO-1^(bright)/VCAM-1⁺ BMMNC showing intense staining of most cellswith anti-collagen type I antibody, (magnified 40×). (D) Lightmicroscopic view of a purified STRO-1^(bright)/VCAM-1⁺, allowed toadhere to fibronectin-coated culture adopts a stellate, fibroblastoidmorphology.

FIG. 4 Characterization of BM MPC. Dual-colour flow cytometric analysisof Ki67 (FITC) expression by STRO-1⁺ (PE) BMMNC isolated by MACS. Thedot plot histogram represents 5×10⁴ events collected as listmode data(B). Telomerase activity in sorted cells populations was examined usinga modified TRAP assay (C). TRAP products derived from CHAPS extracts ofnon-denatured (−) and denatured (+) total bone marrow (lanes 1 and 2),Total STRO-1 [MACS-selected] (lanes 2 and 3). STRO-1^(bright)/VCAM-1⁺cells sorted fraction (lanes 4 and 5), cultured. STRO-1^(bright)/VCAM-1⁺cells (lanes 6 and 7) and CD34⁺-sorted cells TRAP products were resolvedon a 12% polyacrylamide gel, stained with SYBR green fluorescent dye,and visualised using a fluorescence scanning system.

FIG. 5 A total of 44 CFU-F colonies derived from two BM samples wereanalysed for their cumulative production of cells. A marked variation inprolifertive capacity between individual MPC is evident. The majority ofclones ( 36/44; 82%) exhibited only modertate growth potential which didnot persist beyond 12 population doublings. 8/44 clones (18%)demonstrated continued growth extending beyond 17 doublings. All cloneswere switched to adipogenic growth conditions, and under theseconditions, 14/44 clones (32%) exhibited adipogenesis.

FIG. 6 RT-PCR analysis of gene expression in STRO-1^(bright)/VCAM-1⁺purified stromal precursor cells (MPC) isolated directly from marrowaspirates, non-induced primary stromal cultures derived from MPC(CFU-F),and CFU-F cultured under osteogenic-(BONE), chondrogenic-(CART) andadipogenic-(FAT) inductive growth conditions. Various markers of: BONE[transcription factor CBFA1; collagen type I (COLL-I); bonesialoprotein(BSP); osteopontin (OP); osteonectin (ON); osteocalcin (OCN),parathyroid hormone receptor (PTHR)]; FAT [lipoprotein lipase (LPL),transcription factor PPARγ2, leptin, human adipocyte lipid bindingprotein (H-ALBP)]; CARTILAGE [collagen type II (COIL-II), collagen typeX (COLL-X), Aggrecam (AGGN)]. Reaction mixes were subjected toelectrophoresis on a 1.5% agarose gel and visualised by ethidium bromidestaining.

FIG. 7 In vitro developmental potential of MPC. Primary cultures ofderived from STRO-1^(bright)/VCAM-1+ BMMNC were cultured for 2 weeksthen induced under either osteogenic, adipocytic or chondrocyticconditions for 3–5 weeks. A von Kossa positive mineralised matrix formedthroughout the cultures within 4 weeks of bone induction (200×) (A). Thepresence of clusters of lipid containing adipocytes were also detectedby oil red-O staining (200×) (B). Cultures were counter stained withhaematoxylin.

FIG. 8 New bone formation in vivo. ImmunoselectedSTRO-1^(bright)/VCAM-1⁺ BMMNC clones, expanded in vitro, were implantedsubcutaneously into SCID mice using porous ceramic cubes. Implants wereharvested 8 weeks post transplant. New bone formation (solid arrow) wasobserved for a proportion of clones within the cavities of the ceramiccubes (open arrow) together with surrounding fibrous and hematopoietictissue (40×) (A). The sections were counter stained with haematoxylinand eosin. A magnified view of new bone formation is shown depicting anosteocyte (arrow) (200×) (B).

FIG. 9 Dual parameter flow cytometric analysis of STRO-1⁺ human bonemarrow mononuclear cells isolated by MACS. A distinct subpopulation ofSTRO-1^(bri) cells are identified by VCAM-1, THY-1 (CD90), MUC-18(CD-146) and STRO-2.

To properly investigate the biology of BM MPC, studies were designed toisolate MPC from a heterogeneous population of unfractionated BM cells.This was achieved by using a combination of positive immunoselectionprocedures based on the unique specificity of the STRO-1 mab, in orderto maximise the recovery and purity of the MPC population. Following theisolation of homogeneous populations of MPC we then explored theirpattern of gene expression for various bone-, fat- and cartilage-relatedmarkers to determine the degree of commitment towards different stromalcell lineages in vivo. Finally we have investigated the developmentalpotential of purified populations of BM MPC in vitro under definedconditions [Gronthos et al, 1994] and in vivo by ectopic implantationinto immunodeficient mice [Haynesworth et al, 1992].

We and others have had success in isolating MPC based on theirexpression of the STRO-1 antigen either by FACS or by usingimmunomagnetic particles, such as Dynabeads [Tamayo et al, 1994] or bymagnetic-activated cell sorting (MACS) [Gronthos et al, 1995 and 1998].The latter was used initially to provide a reproducible technique forisolating BM derived MPC with the capacity to process high cell numbers.The mab STRO-1 proved to be an ideal reagent for isolating MPC fromadult BM because of its lack of reactivity to haemopoietic progenitors[Simmons and Torok-Storb, 1991a] yielding a clean separation between MPCand haemopoietic progenitors in adult BM. Moreover, the antigenidentified by STRO-1 was found in the present study to be expressed atparticularly high copy number by MPC, which may in part account for thehigh efficiency and recovery of BM CFU-F observed. These studiesidentified the minor STRO-1^(bright) subset of the total STRO-1⁺ BMMNCfraction to contain the CFU-F population. However the resulting postMACS STRO-1^(bright) cell population was only partially enriched forMPC.

We have previously demonstrated that the cell surface antigen, VCAM-1 isuniversally expressed on BM MPC and their progeny [Simmons et al, 1992,1994]. This is in contrast to other markers expressed by BM MPC such asTHY-1, CD10, CD13, and thrombomodulin, [Simmons et al, 1994] which arealso known to react with either haemopoietic cells and or platelets[Baum et al, 1992; Conway and Nowakowski, 1993; Ship and Look, 1993].The VCAM-1 molecule is a transmembrane glycoprotein with a molecularweight of between 95 and 110 kDa present on the membranes of stromalcells and endothelial cells [Osborn et al, 1989; Simmons et al, 1992].The immunoglobulin super family member is one ligand for the integrinreceptor α4β1 (VLA-4) present on haemopoietic stem cells, and isinvolved in the recruitment of lymphocytes and monocytes expressing α4β1to sites of infection and inflammation [Elices et al, 1990; Simmons etal, 1992]. Significantly, VCAM-1 only reacted with a minor proportion ofBMMNC effectively subletting the total STRO-1⁺ population, reactingpreferentially with the STRO-1^(bright) cell fraction. The BM MPCpopulation was subsequently shown to reside exclusively in theSTRO-1^(bright)/VCAM-1⁺ fraction of human adult BM.

The absolute frequency of MPC in bone marrow was determined by limitingdilution experiments using Poisson distribution statistics. Otherstudies using this statistical analysis have shown that murine BMosteoprogenitor cells with the potential to form mineralized bonenodules in vitro, occurred at a frequency of 1 per 1000 BM cells plated,based on the phenotype 5-fluoracil resistant, haemopoietic lineagemarker negative [Van Vlasselaer, 1994]. These osteoprogenitorsrepresented approximately 20% of the total MPC population in normalmurine BM [Falla et al, 1993; Van Vlasselaer, 1994]. Similar analyses offetal human BMMNC demonstrated the frequency of MPC at 1 per 1,000 to 1per 100,000 cells plated, at 14 weeks and 24 weeks gestation,respectively, based on the immunophenotype CD34⁺/CD38⁻/HLA-DR⁻ [Walleret al, 1995a]. Furthermore, additional subletting of fetal BM using thehaemopoietic marker CD50, distinguished HSC from the MPC population, butfound no significant difference in the incidence of clonogenic stromalcells sorted on the basis of the phenotype CD34⁺/CD38⁻/HLA-DR⁻/CD50⁻[Waller et al, 1995b]. However, no stromal progenitors were observedwhen single cells of human adult BM samples were sorted based on theCD34⁺/CD38⁻/HLA-DR⁻ phenotype [Waller et al, 1995a]. This may be due tothe inefficiency of a predominantly negative selection criteria used toisolate fetal BM MPC and may also reflect the use of the CD34 antigenwhich demonstrates low level expression on adult BM MPC [Simmons andTorok-Storb, 1991b].

In the illustrated embodiment, the incidence of clonogenic cells(clusters 10<50 cells+colonies 50) from adult human BM was determined tobe 1 per 2 STRO-1^(bright)/VCAM-1⁺ cells plated in SDM containing PDGFand EGF. Using serum-deprived medium significantly enhances theincidence of clonogenic growth over that of serum replete cultures,particularly at low plating densities [Gronthos and Simmons, 1995]. Itmust also be stated that a proportion of the wells which were scored as‘negative’ contained cell clusters of less than 10 cells. Therefore, byfurther refining the CFU-F culture assay, it may be possible tostimulate the growth of MPC in order to increase the overall purity ofthe MPC population based on the composite STRO-1^(bright)/VCAM-1⁺phenotype. Nevertheless, the combined purification technique of theillustrated embodiment effectively achieved a several thousand foldincrease in the incidence of BM MPC when compared to unfractionatedBMMNC.

The cells contained within the STRO-1^(bright)/VCAM-1⁺ BM fraction werefound to be a homogeneous population of large cells with extensivecytoplasmic processes existing in vivo in a non-cycling state. Otherstudies have found that MPC residing in the BM are almost entirelynon-cycling as shown by ³H thymidine labelling in rodents and by meansof the in vitro thymidine suicide technique in humans [Castro-Malaspinaet al, 1980; Castro-Malaspina et at, 1981]. This data coincides with theobservations that primitive multi-potential stem cells, identified inthe other cell systems such as HSC are by definition quiescent cells[Andrews et al, 1986; Szilvassy et al, 1989; Li and Johnson, 1992].Given the reported developmental potential of cultured BM MPC in vitroand in vivo the question arises as to whether these cells are trulyrepresentative of an early uncommitted phenotype with multi-potential orwhether all or a proportion of the CFU-F are already committed towards aparticular stromal cell lineage.

Analysis of the gene expression pattern of purified adult BM MPC in theillustrated embodiment has revealed that many of the genes expressed byCFU-F in vivo demonstrate a broad stromal tissue distribution related toosteoblasts, adipocytes and chondrocytes. It is very common to find inthe literature that many markers for example osteonectin, osteopontin,and alkaline phosphatase in the bone cell lineage are described as beingspecific to bone cells, when in fact these markers have a wider tissuedistribution. Therefore, it is not surprising to find that MPCidentified by STRO-1 share common markers with differentiated stromalcell types. Importantly, specific markers of commitment such as CBFA-1,collagen type II, PPARγ2, [reviewed in Rodan and Harada, 1997] to bone,cartilage and fat respectively were not expressed by theSTRO-1^(bright)/VCAM-1⁺ population in fresh BM aspirates. In addition,immunohistochemical examination of STRO-1^(bright)/VCAM-1⁺ sorted BMMNCfailed to show any reactivity to the smooth muscle marker α-smoothmuscle actin or with the endothelial marker, FVIII. Therefore the MPCresiding in the BM seem to exist in an uncommitted state, and may havethe potential under different conditions to develop into a few or all ofthe stromal elements recognised in the bone marrow microenvironment.

In the present study, cultures of purified STRO-1^(bright)/VCAM-1⁺ humanBM CFU-F typically developed a von Kossa positive mineral by twenty onedays under osteogenic conditions (ASC-2P, PO_(4i), DEX). The presence ofmineral deposits was demonstrated in all CFU-F clones examined, where40% of the clones also displayed the capacity to differentiate intoadipocytic cell clusters. Moreover, individual CFU-F clones were alsofound to contain a small proportion of fibroblastic-like cells notassociated with either mineralization or lipid accumulation. Thesefibroblast-like cells may represent as yet undefined stromal populationssuch as reticular cells, smooth muscle cells, bone lining cells,osteocytes and committed stromal progenitors.

The developmental potential of selected CFU-F clones was furtherexamined in vivo. The porous hydroxyapatite coated ceramic cubesreproducibly supported the development of human osteogenic tissue inSCID mouse. This is in agreement with the findings in previous in vivostudies using unfractionated rodent and human BM mesenchymal cellcultures [Haynesworth et al, 1992a; Krebsbach et al, 1997; Kusnetsov etal, 1997]. In the present study, pretreating the HA ceramic cubes withpurified fibronectin was critical to maximise the number of cellsretained in the cubes after loading prior to transplantation (data notshown). Pre-treatment of HA cubes with fibronectin and laminin has beenreported to increase cell retention and spreading on the ceramic surfaceof the cubes [Dennis et al, 1992; Dennis and Caplan; 1993]. Fibronectinand laminin coated cubes were found to augment bone formation fromcultured rat BM mesenchymal cells at earlier time points in comparisonto untreated cubes [Dennis et al, 1992; Dennis and Caplan, 1993].

The present study failed to detect cartilage formation in any of thetransplantation models used, in contrast to other studies whichdemonstrated cartilage formation in diffusion chambers transplanted withrodent bone marrow or mesenchymal cells derived from the marrow of youngchildren. To date, there have been no reports describing thereproducible induction of cartilage formation using adult human bonemarrow stromal cells in vivo or in vitro. In the present study, theexpression of the hypertrophic chondrocyte marker collagen type X, bypurified adult human BM MPC, is somewhat puzzling, given the presumedspecificity of this molecule. Since the physiological role of collagentype X is unknown, its significance in bone marrow remains to bedetermined.

The present work is in accord with previous studies showing that theformation of new bone in implants of HA cubes is attributed todifferentiation of human mesenchymal cells into functional osteoblasts[Kusnetsov et al, 1997] and did not result from the recruitment ofosteoprogenitors from the surrounding host (mouse) tissue. Furthermore,other cell types present such as muscle, adipocytes and vascularendothelial cells showed no hybridization with the alu probe and aretherefore presumed to be host in origin. These findings demonstrate thata proportion of BM MPC within the STRO-1^(bright)/VCAM-1⁺ BMsubfraction, demonstrate the capacity to develop into multiple stromalcell types including osteoblasts, adipocytes and fibroblast-like cells.

Further subletting of the STRO-1^(bright)/VCAM-1⁺ BM fraction usingthree- and four-colour FACS analysis may eventually provide a means todiscriminate between subpopulations contained within the MPC pool whichexhibit different developmental potentials. The purification of MPCclones with different potential may then be used to generatemultipotent, bi-potent and uni-potent cell lines which could greatlyfacilitate the design of experimental approaches to study the molecularmechanisms regulating the commitment of early precursors into differentstromal cell lineages.

One area of potential benefit that will occur from a greaterunderstanding of the proliferation and differentiation of MPC, is theability to manipulate and expand mesenchymal cell populations in vitrofor subsequent reimplantation in vivo. The use of animal models hasdemonstrated the efficacy of utilising ex vivo expanded BM mesenchymalcells to facilitate bone regeneration and tendon repair in vivo [Bruderet al, 1998b; 1998c; Young et al, 1998]. Several studies have alsodescribed how cultured marrow stromal cells from a variety of speciesare readily infected using either amphotropic retroviruses oradenoviruses [Harigaya and Handa, 1985; Rothstein et al, 1985; Singer etal. 1987; Cicutinni et al, 1992; Roecklein and Torok-Storb, 1995]. Inaddition, some studies have demonstrated the persistence of transplantedtransduced cells over several months in animal models [Li et al, 1995;Anklesaria et al, 1996; Onyia et al, 1998 Reiw et al, 1998]. Thereforethe ability to harvest purified human MPC from aspirates of BM and toexpand these cells ex vivo makes them ideal candidates as possiblevehicles for gene transfer, in order to treat a variety of diseases andgenetic disorders.

Materials and Methods

Subjects

Aspirates of human BM samples were obtained from the iliac crest and thesternum of normal adult volunteers with their informed consent,according to procedures approved by the ethics committee at the RoyalAdelaide Hospital, South Australia. Bone marrow mononuclear cells(BMMNC) were obtained by centrifugation over Ficoll 1.077 g/ml(Lymphoprep, Nycomed, Oslo, Norway) at 400 g for 30 minutes (min) andthen washed and resuspended with Hank's buffered saline solutioncontaining 1% bovine serum albumin and 10 mM HEPES, pH 7.35 (HBSS).

Isolation of STRO-1+ Cells by Magnetic-Activated Cell Sorting (MACS)

This procedure is a modification of that described elsewhere [Gronthoset al, 1998]. Approximately 1×10⁸ BMMNC were incubated with STRO-1supernatant for 60 min on ice. The cells were then washed in HBSS andresuspended in 1 ml of HBSS containing a 1/50 dilution of biotinylatedgoat anti-mouse IgM (μ-chain specific; Southern BiotechnologyAssociates, Birmingham, Ala.) for 45 min on ice. Following this, thecells were washed twice in MACS buffer (single strength Ca²⁺ and Mn²⁺free PBS supplemented with 1% BSA, 5 mM EDTA and 0.01% sodium azide) andresuspended in 900 μl of MACS buffer to which 100 μl of streptavidinmicrobeads (Miltenyi Biotec, Bergisch Gladbach, F.R.G.) was added. Thecells were further incubated for 15 min on ice after whichstreptavidin-fluorescein isothiosyanate (FITC) conjugate ( 1/50; CaltagLaboratories, San Francisco, Calif.) was added directly to thesuspension for an additional 5 min. The cells were separated on a MiniMACS magnetic column (column capacity 10⁷ cells, Miltenyi Biotec)according to the manufacturers specifications.

Purification of the CFU-F Population by Fluorescence Activated CellSorting (FACS)

Dual colour-FACS analysis of the STRO-1^(bright) population was achievedby incubating the MACS isolated STRO-1 population with saturating levelsof the Mab 6G10 (mouse IgG1 anti-human CD106: vascular endothelialadhesion molecule-1, VCAM-1; kindly donated by Dr. B. Masinovski FCOSCorp., Seattle Wash.) for 30 min on ice. After washing with HBSS thecells were incubated with a second label goat anti-mouse IgG (γ-chainspecific) phycoerythrin (PE) conjugate antibody ( 1/50; SouthernBiotechnology Associates, Birmingham, Ala.) and a streptavidin-FITCconjugate ( 1/50; CALTAG Laboratories, San Francisco, Calif.) for 20 minon ice. The cells were then washed in HBSS prior to being sorted usingthe automated cell deposition unit (ACDU) of a FACStar^(PLUS) (BectonDickinson, Sunnyvale, Calif.) flow cytometer. STRO-1^(bright)/VCAM-1+cells were seeded at plating densities of 1, 2, 3, 4, 5, and 10 cellsper well (96-well plates) in replicates of 24 wells per plating density(FIG. 2). The cells were cultured in serum deprived medium onfibronectin coated wells as previously described [Gronthos and Simmons1995; Gronthos et al, 1998]. On day 10 of culture the cells were thenfixed and stained for 60 min with 0.1% toluidine blue in 1%paraformaldehyde. Aggregates of 50 cells were scored as CFU-F coloniesand aggregates of 10<50 cells were scored as clusters using an OlympusSZ-PT dissecting light microscope (Olympus Optical Co. Ltd. Tokyo,Japan).

Analysis of Cell Cycling Status of STRO-1+ BMMNC

The STRO-1⁺ BMMNC were isolated by MACS as described above and thenincubated with streptavidin PE for 15 min on ice. After washing twicewith PBS the cells were fixed for 10 min with cold methanol (70%) onice. Following this, the cells were washed three times with PBS and thenincubated in blocking buffer for 15 minutes. The monoclonal antibodyKi-67 conjugated to FITC (DAKOPATTS A/S, Glostrup, Denmark) was addeddirectly to the cells ( 1/10 dilution) in blocking buffer for 45 min onice served as the negative control.

RNA Isolation and First-Strand cDNA Synthesis

Total cellular RNA was routinely prepared from 2×10⁴STRO-1^(bright)/VCAM-1⁺ cells collected as a bulk population and lysedusing RNAzolB extraction method (Biotecx Lab. Inc., Houston, Tex.), asper manufacturers recommendations. RNA isolated from each subpopulationwas then used as a template for cDNA synthesis. cDNA was prepared usinga First-strand cDNA synthesis kit from Pharmacia Biotech (Uppsala,Sweden) according to manufacturers instructions. Briefly, total RNA wasresuspended in 8 μl of DEPC-treated water and subsequently heated to 65°C. for 10 min. Following snap cooling on ice, the RNA was added to 7 μlof premix containing reaction buffer, oligo-dT as primer and SuperscriptMMLV Reverse transriptase. Following incubation at 42° C. for 60 min,the volume of the reaction was adjusted to 50 μl with the addition of 35μl of sterile water. The samples were stored at −20° C.

Polymerase Chain Reaction (PCR)

Due to limiting cell numbers, the expression of various bone-relatedtranscripts (Table I) was assessed by polymerase chain reaction (PCR)amplification, using a standard protocol [Sambrook et al. 1989]. Twomicroliters of first strand cDNA mixture from each subpopulation wasdiluted in a 50 μl PCR reaction (67 mM Tris HCI pH 8.8, 16.6 mM(NH₄)₂SO₄, 0.45% Triton X100, 200 μg/ml gelatin, 2 mM MgCl₂, 200 μM eachdNTP) containing 100 ng of each primer (Table 1), to which 2.5 units ofAmplitaq DNA Polymerase (Perkin-Elmer, Norwalk, Conn., USA) was added.Reaction mixes were overlayed with mineral oil and amplificationachieved by incubation in a Perkin-Elmer/Cetus thermal cycler. Primerdesign enabled typical cycling conditions of 94° C./(2 min), 60° C./(30sec), 72° C./(1 min) for 40 cycles, with a final 10 min incubation at72° C. To control for the integrity of the various RNA preparations, theexpression of GAPDH and/or beta-2-microglobulin was also assessed.Following amplification, 10 μl of each reaction mixture was analysed by1.5% agarose gel electrophoresis, and visualised by ethidium bromidestaining.

The Developmental Potential of BM CFU-F In Vitro

We have previously reported the conditions for the induction of humanbone marrow stromal cells to develop a mineralised bone matrix in vitro[Gronthos et al, 1994]. Briefly, the osteogenic and adipocytic potentialof thirty day 4 CFU-F clones derived from single STRO-1^(bright)/VCAM-1⁺sorted cells was assessed by culturing in alpha modification of Eagle'smedium (α-MEM: Flow Laboratories) supplemented with 20% FCS, L-glutamine(2 mM), β-mercaptoethanol (5×10⁻⁵ M), L-ascorbic acid 2-phosphate (100pM) (ASC-2P: Novachem, Melbourne, Australia), dexamethasone sodiumphosphate (10⁻⁸ M) (DEX: David Bull Laboratories, Sydney, Australia),KH₂PO₄ (1.8 mM) (BDH Chemicals) and Hepes (10 mM), at 37° C., 5% CO₂.The media was changed twice a week for a period of six weeks. Cultureswere rinsed twice with PBS then fixed in situ with 10% neutral formalinfor 30 mon. Staining for vonKossa was performed according to the methodof Pearse and Gardner (1972). Sections or culture wells were washedtwice in distilled water and then stained in 5% aqueous AgNO₃ for 60 minunder ultraviolet light. After staining with AgNO₃, the sections werewashed twice with distilled water and then placed in 5% sodiumthiosulphate for 1 min. Cultures were washed in distilled water, counterstained with Mayer's haematoxylin and mounted. Oil Red O (ORO) stainingwas performed as described by Grimble (1998). Briefly, cultures werefixed as described above, washed twice with PBS and air dried. Cultureswere immersed in a solution 0.5% (w/w) ORO in isopropanol for 15 min atroom temp., washed three times with distilled water and subsequentlycounterstained with haematoxylin.

Similarly, the chondrogenic potential of the same clones was assessed byculturing 2×10⁵ cells per clone in 0.5 mls SDM supplemented with TGFβ1and gently centrifuged at 200 g for 2 min in a 10 ml polypropolene tubethen incubated at 37° C., 5% CO₂. The media was changed twice a week fora period of three weeks

The Developmental Potential of BM CFU-F In Vivo

Bulk cultures of CFU-F derived from STRO-1^(bright)/VCAM-1⁺ sorted BMMNCwere cultured for 5 weeks in the presence of ASC-2P and DEX and 10% FCS.The adherent cell layers were trypsinised and seeded onto 27 mm³ poroushydroxyapatite ceramic cubes (Zimmer Corporation, Warsaw, Ind., USA)pre-coated with fibronectin (5 μg/ml) (Boehringer Mannheim, Germany).The ceramic cubes were then implanted into subcutaneous pockets into thebacks of SCID mice for a period of up to 8 weeks as described previously[Haynesworth et al, 1994; Kuznetsov et al, 1997]. Recovered implantswere fixed in 10% buffered formalin for 2 days then decalcified for afurther seven days in 0.5 M EDTA before being embedded in paraffin wax.Cross-sections of the cubes were prepared as 5 μm sections onto glassslides pre-coated with Cell-Tak and counter stained with haematoxylinand eosin.

In Situ Hybridization for the Human Specific Alu Sequence

The HA ceramic implants were recovered 8 weeks post transplant andprepared for paraffin embedding on Cell-Tak coated slides as describedabove. To determine the origin of the cells within the implants in situhybridization analysis was performed using a DNA probe specific to theunique human repetitive alu sequence [Kuznetsov et al, 1997]. The humanspecific alu sequence (pBLUR8; ATCC) was subcloned into the BamH1restriction site of a pGEM-4Z plasmid (Promega). Thedigoxigennin-labeled alu specific probe was prepared by PCR containing1×PCR buffer (67 mM Tris HCl pH 8.8, 16.6 mM (NH₄)₂SO₄, 0.5%Triton-X100, 0.2 μg/ml gelatin, 2.5 mM MgCl₂, 0.2 mM dATP, 0.2 mM dCTP,0.2 mM dGTP, 1.9 mM dTTP, 0.1 mM digoxygenin-11-dUTP (BoehringerMannheim), and 0.25 units of Amplitaq DNA Polymerase) and 100 ng of SP6and T7 primers (Table 1) and 1 ng of plasmid DNA (pGEM-4Z; PromegaCorp., Madison, Wis.) containing the alu sequence subcloned into theBamHI restriction site from (pBLUR8; ATCC, Rockville, Md.). Sectionswere deparaffinized with xylene and ethanol then rehydrated throughgraded (100%, 90%, 70%, 50%) ethanol solutions. The sections were thentreated with 0.2 N for 7 min at room temperature and then incubated in 1mg/ml pepsin (Sigma, St. Louis, Mo.) in 0.1 N HCl for 10 minutes at 37°C. After washing in PBS, the sections were treated with 0.25% aceticacid containing 0.1 M triethanolamine (pH 8.0) for 10 min andprehybridized with 50% deionized formamide containing 4×SSC for 15 minat 37° C. The hybridization solution (1 ng/μl digoxigenin-labeled probein 1× Denhardt's solution, 5% dextrane sulfate, 0.2 mg/ml, salmon spermDNA, 4×SSC, 50-% deionized formamide) was then added to the sections fordenaturation at 95° C. for 3 minutes followed by hybridization at 45° C.for 3 hr. After washing with 2×SSC and 0.1×SSC, digoxigenin-labeled DNAwas detected by immunohistochemistry using antidigoxigenin alkalinephosphatase-conjugated Fab fragments ( 1/5000; Boehringer MannheimCorp., GMBH, Germany) followed by incubation with the correspondingalkaline phosphatase nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate solution asrecommended by Boehringer Mannheim. Micrographs were taken withEktachrome 64 T colour film using an Olympus IMT-2 inverted lightmicroscope.

Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase activity was measured by a modified non-radioactive TRAPprotocol essentially as described by Fong et al (1997). Telomerase cellextracts were prepared by the method of Kim et al, (1994), with minormodifications. Populations of sorted or cultured cells were lysed inice-cold CHAPS extraction buffer (0.5%3[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate], 50 mMTris-HCI, pH 7.4, 5 mM MgCl₂, 5 mM EGTA, 25 mM 2-mercaptoethanol, 1ng/ml leupeptin, and 50% glycerol in DEPC-treated water), at aconcentration of 1000 cells/μl, incubated on ice for 30 minutes andcentrifuged at 16000×g for 20 minutes at 4° C., the supernatantrecovered and stored at −80° C. until required. Detection of telomeraseactivity was performed in a two-step process as previously described(Fong et al, 1997). Briefly, to 2 μl of cell extract, 16.5 μl of TRAPreaction buffer (20 mM Tris-HCI, pH8.2, 1.5 mM MgCl₂, 63 mM KCl, 0.05%Tween-20, 1 mM EGTA), 100 ng of each of TS primer(5′-AATCCGTCGAGCAGAGTT-3′, SEQ ID NO: 1), and CX-ext primer(5′-GTGCCCTTCCCTTACCCTTACCC TAA-3′, SEQ ID NO: 2), 0.5 μL dNTPs (10 mMstock) were added, and the reaction mix incubated at 25° C. for 30minutes. Telomerase was subsequently inactivated by heating the reactionto 90° C. for 2 minutes, prior to the addition of 5 μl of PCR mixture,containing 3.5 μl of TRAP reaction buffer, 1 μl of CX-ext primer and 2.5U Taq polymerase. Reaction mixes were covered with mineral oil andplaced in a Hybaid thermocycler, and subjected for 34 cycles of 94° C.for 30 seconds, 50° C. for 30 seconds and 72° C. for 45 seconds, with afinal extension at 72° C. for 2 minutes. To confirm the specificity ofthe telomerase products, in all cases, a 2 μl aliquot of each CHAPSlysate was subjected to denaturation by heating samples at 100° C. for10 minutes. 25 μl of each reaction was resolved on a non-denaturing 12%polyacryalmide gel, and visualised by staining width SYBR Greenfluorescent dye (FMC Bioproducts, OR, USA) as recommended by themanufacturer. The TRAP products were analysed using a fluorescencescanning system (Molecular Dynamics, Sunnyvale, Calif., USA).

Transmission Electron Microscopy (TEM)

STRO-1^(bright)/VCAM-1⁺ cells (approximately 2×10⁴ cells) were collectedas a bulk population into eppendorf microtubes, washed once in 0.05 Msodium cacodylate buffer and then fixed in 2.5% glutaraldehyde (EMGrade) in cacodylate buffer for 2 hr. The cultures were postfixed with2% osmium tetroxide (VIII) (BDH Chemicals) in cacodylate buffer for 1hr. After this, the cultures were dehydrated with graded ethanolsolutions (70%, 90%, 100%). Epoxy resin (TAAB Laboratories; Berkshire,England) was then used to infiltrate the cultures overnight at 37° C.Polymerization of the resin was carried out at 60° C. for 24 hr undervacuum. Ultrathin sections were cut on a LKB 8800 Ultrotome II (Broma,UK) and mounted onto copper grids. Sections were then examined using aJEOL 1200 EX II (Tokyo, Japan) transmission electron microscope.Photographs were taken using ILFORD EM Technical film.

Results

Isolation and Purification of STRO-1⁺ BM MPC

We have previously demonstrated the effectiveness of MACS to isolate andenrich for MPC from aspirates of human BM based on the cell surfaceexpression of the STRO-1 antibody [Gronthos and Simmons, 1995; Gronthoset al, 1998]. In the present study, flow cytometric analysis of MACSisolated STRO-1⁺ BMMNC cells demonstrated a heterogeneous pattern ofexpression spanning over four logs in fluorescence intensity (FIG. 1).Single-color FACS was subsequently employed to sort the STRO-1⁺ BMMNCfraction into three subsets; STRO-1^(dull) STRO-1^(intermediate) andSTRO-1^(bright). Clonogenic assay for CFU-F in the different sortedSTRO-1⁺ subpopulations demonstrated that the majority of the MPC werecontained within the STRO-1^(bright) cell fraction. There was a 900 foldincrease in the incidence of CFU-F in the STRO-1^(bright) populationwhen compared to unfractionated BMMNC (Table 1) demonstrating that BMMPC contained a high copy number of the STRO-1 antigen on their cellsurface. The recovery of the MPC population in the STRO-1^(bright)fraction was >75% in respect to the estimated total number of CFU-F inthe BM sample pre-MACS.

We attempted to obtain a more accurate discrimination of theSTRO-1^(bright) subset by incubating the total STRO-1⁺ MACS isolatedcells with the stromal cell surface antigen VCAM-1 (FIG. 2A) previouslyfound to react exclusively with BM MPC [Simmons et al, 1994]. Dualcolor-FACS was used to identify and isolate the STRO-1^(bright)/VCAM-1⁺BMMNC fraction. Limiting dilution analysis was subsequently performed,using the FACStar^(PLUS) automated cell deposition unit, to seedSTRO-1^(bright)/VCAM-1⁺ cells at various plating densities as describedin the methods. Cells were cultured under serum deprived conditions inthe presence of PDGF and EGF (10 ng/ml) previously found to support theclonogenic growth of CFU-F above that of serum replete conditionsparticularly at low plating densities [Gronthos and Simmons, 1995]. Themean incidence (n=6 different BM donors) of day 10 CFU-F colonies (>50cells) was determined to be 1 CFU-F per 3 STRO-1^(bright)/VCAM-1⁺ cellsplated using Poisson distribution statistics (FIG. 2B). Furthermore, theincidence of clonogenic cells (clusters >10<50 cells+colonies) was foundto be 1 per 2 STRO-1^(bright)/VCAM-1⁺ cells plated (FIG. 2C). TheMACS/FACS purlfication technique effectively achieved a 5×10³ foldenrichment of the CFU-F population when compared to unfractionated BMMNCwith an average incidence of 1 CFU-F colony per 10⁴ BMMNC. It must alsobe stated that a proportion of the wells which were scored as ‘negative’contained cell clusters of less than 10 cells.

Characterization of Purified BM MPC

Morphological examination of freshly sorted STRO-1^(bright)/VCAM-1⁺cells was carried out by transmission electron microscopy. Purified BMCFU-F appeared to be a homogeneous population of large cells containingmany cytoplasmic processes and a large nucleous with an open chromatinstructure (FIG. 3). To determine the cell cycling status of the CFU-Fpopulation in aspirates of BM the MACS isolated STRO-1⁺ BMMNC fractionwas further incubated with the cell cycling specific antigen Ki-67[Gerdes et al, 1984; Wersto et al, 1988]. Two color flow cytometricanalysis revealed that the STRO-1^(bright) subset which contained theCFU-F population lacked co-expression of the Ki-67 antigen demonstratingthat these cells are non-dividing in vivo (FIG. 4A). Telomerase activitywas examined in cell extracts from sorted and cultured candidate stromalprogenitor cell populations by a modified TRAP assay. Telomeraseactivity was present in all fractions including the candidate stromalstem cell compartment isolated from adult bone marrow, defined by theirexpression of both the STRO-1 and VCAM-1 (CD106) cell surface molecules(FIG. 4B).

To assess the proliferative capacity of BM MPC, individual CFU-Fcolonies (n=44) derived from two BM samples were expanded in thepresence of serum under normal clonogenic growth conditions. A minorproportion of clones ( 8/44, 18%) demonstrated continued growthextending beyond 20 population doublings while the remainder showedlittle or no proliferation beyond 12 population doublings (FIG. 5).These cells also appeared to be capable of differentiating into adiposecells, whereas other isolated cells were less likely to do so.

A detailed phenotypic analysis of freshly isolated BM MPC pre-culturewas compiled. Total RNA obtained from STRO-1^(bright)/VCAM-1⁺ cells wasused to generate full-length first-strand cDNA as described in themethods. RT-PCR analysis revealed the presence of various bone cellmarkers including bonesialoprotein, osteonectin, and collagen type I.However, there was an absence in the expression of osteopontin, theparathyroid hormone receptor, and the more specific bone cell markersosteocalcin and the transcription factor CBFAI (FIG. 6A). Similarly, thefat-related markers lipoprotein lipase and the adipocyte human lipidbinding protein were found to be expressed by theSTRO-1^(bright)/VCAM-1⁺ population but there was no detectableexpression of the adipocyte specific markers, the obese gene product(leptin) and the early transcription factor PPARγ2 in these cells (FIG.6B). Furthermore the cartilage specific markers collagen type II andaggrecan were also not expressed by our purified MPC population. Howeverthe STRO-1^(bright)/VCAM-1⁺ cell fraction was found to express collagentype X, a marker associated with hypertrophic chondrocytes (FIG. 6C). Inaddition, cytospin preparations of STRO-1^(bright)/VCAM-1⁺ sorted BMMNCfailed to show any reactivity to the smooth muscle marker α-smoothmuscle actin or with the endothelial marker, FVIII (data not shown).Overall the MPC population appeared to represent an early precursorpopulation not yet fully commited to anyone particular stromal celllineage.

Culture expanded bulk CFU-F derived from STRO-1^(bright)/VCAM-1⁺ sortedcells were assessed for their ability to develop into functionalosteoblasts, chondrocytes and adipocytes in vitro as previouslydescribed [Gronthos et al, 1994]. A von Kossa positive mineralisedmatrix developed throughout the cultures by the end of the sixth week ofinduction (FIG. 7A). In addition, clusters of Oil Red O positiveadipocytes were observed within the adherent layers in the same cultures(FIG. 7B). Following three weeks of chondrocytic induction in thepresence of TGFβ1, the cells were also found to express the cartilagespecific marker collagen type II by immunohistochemistry. FurthermoreRT-PCR analysis of total RNA isolated from the different cultureconditions demonstrated the expression of markers specific to bone(CBFA-1, OCN, PTH-R), fat (PPARγ2, leptin) and cartilage (collagen typeII, aggrecan) (FIG. 6B).

The Developmental Potential of BM MPC Clones In Vitro and In Vivo

Bone marrow CFU-F clones were established from STRO-1^(bright)/VCAM-1⁺sorted cells from three individual BM donors. At day 4 of culture,single clonogenic clusters were identified and expanded by subculture.Half of the cells from the first passage were taken from each clone andcultured under osteogenic growth conditions as described above. Theosteogenic potential of ninety CFU-F clones was assessed where a vonKossa positive mineralised matrix formed in all of the ninety clones.However, only a proportion (38% ±15 SEM, n=3) of the same clones gaverise to clusters of lipid containing oil red-O positive adipocytesdemonstrating the bi-potential of the CFU-F population in vitro.

Half the cells from a representative 46 clones were subcultured andexpanded for several weeks, then seeded into porous HA ceramic cubes andimplanted subcutaneously into SCID mice for a period of 8 weeks aspreviously described [Haynesworth et al, 1992, Kusnetsov et al, 1997].Cross-sections of the cubes prepared for histiological examinationshowed that all of the implants contained an extensive network of bloodvessels and fibrous tissue (FIG. 8A and FIG. 8B). Bone formation wasfound in 42% (n=26) and 55% (n=20) of the clones isolated from twodifferent BM aspirates. The ability of individual MPC clones to form avon Kossa positive mineralised matrix in vitro did not always correlateto the development of new bone in vivo. Similarly, the capacity of MPCclones to form adipocytic clusters in vitro had no bearing on thedevelopment of new bone formation in vivo.

The origin of the cellular material within the recovered implants wasassessed by in situ hybridization using a DNA probe specific to theunique human repetitive alu sequence. The fibrous tissue, bone liningcells and osteocytes within the newly formed bone were all found to bepositive for the alu sequence confirming their human origin and thebi-potential of a proportion of BM MPC (FIG. 9C and FIG. 9D).Conversely, the fat and smooth muscle surrounding the ceramic cubes didnot express the alu sequence and was therefore presumed to haveoriginated from the host. Similarly, the endothelium lining the smallblood vessels were also negative for the alu sequence implying they werederived from the mouse vasculature. In addition, there was no cartilageformation observed in sections of different implants and at differenttime points, as assessed by immunohistochemical analysis using apolyclonal antibody specific for collagen type II (data not shown).

Uses of MPCs

EXAMPLE 1 Repair of Articular Cartilage

Damaged articular cartilage generated by trauma or by diseases such asosteoarthritis and rheumatoid arthritis usually does not heal. Howeverit is expected that this type of defect could be treated by implantingcultured MPCs of the present invention into the defect. The carrier maybe pliable to mould to the shape of the defect and to promote round cellshape which is important for induction of chondrocyte differentiation. Asuitable carrier may be constructed of collagen or fibrin. See Caplan etal. in U.S. Pat. No. 5,226,914.

EXAMPLE 2 Repair of Bone

A combination of MPCs as well as a suitable support can be introducedinto a site requiring bone formation. Cultured MPCs contained in calciumphosphate ceramic vehicles may be implanted into the defect site. Forappropriate methods and techniques see Caplan et al. in U.S. Pat. No.5,226,914 and U.S. Pat. No. 5,837,539.

EXAMPLE 3 Anchoring of Prosthetic Devices

The surface of a prosthetic device can be coated with MPCs prior toimplantation. The MSCs can then differentiate into osteogenic cells tothereby speed up the process of bony ingrowth and incorporation of theprosthetic device. See Caplan et al. in U.S. Pat. No. 5,226,914 and U.S.Pat. No. 5,837,539.

EXAMPLE 4 Gene Therapy

An exogenous nucleic acid that encodes a protein or peptide withtherapeutic may be transformed into the enriched population usingstandard techniques (see U.S. Pat. No. 5,591,625 by Gerson et al.). Thetransformed cell population can then be introduced into the body of thepatient to treat a disease or condition. For example, can be used toprovide a continuous delivery of insulin, or genes encoding Factor VIIIwhich is involved in clotting and therefore may be used inhaemophiliacs.

EXAMPLE 5 Marrow Transplantation

A composition containing purified MPCs can be injected into a patientundergoing marrow transplantation prior to the introduction of the wholemarrow. In this way the rate of haemopoiesis may be increased,particularly following radiation or chemotherapy. The composition mightalso include haemopoietic cells for use in radiotherapy or chemotherapy.

REFERENCES

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1. A population of cells enriched for STRO-1^(bright) cells, whereinsuch STRO-1^(bright) cells are mesenchymal precursor cells whichcomprise mesenchymal precursor cells capable of giving rise to colonyforming unit-fibroblasts (CFU-F).
 2. The enriched population of cells asin claim 1, wherein the mesenchymal precursor cells carry at least oneadditional marker selected from the group of surface markers consistingof THY-1, VCAM-1, STRO-2, and CD146.
 3. The enriched population of cellsas in claim 2, wherein the mesenchymal precursor cells carry the markersSTRO-1 and VCAM-1.
 4. The enriched population of cells as in claim 1,wherein said STRO-1^(bright) cells are capable of differentiation intoat least two committed cell types selected from the group consisting ofadipose, areolar, osseous, cartilaginous, elastic, and fibrousconnective tissue.
 5. The enriched population of cells as in claim 1,wherein the enriched population is suitable for seeding onto a vehiclefor implantation to assist in bone growth.
 6. The enriched population ofcells as in claim 1, wherein said STRO-1^(bright) cells in the enrichedpopulation comprise an exogenous nucleic acid that expresses atherapeutic agent transformed into them such that the population ofcells may be introduced into the body of a patient to release thetherapeutic agent.
 7. The enriched population of cells as in claim 1,wherein the enriched population is used to augment bone marrowtransplantation.
 8. A composition comprising the enriched population ofcells of claim
 1. 9. The composition as in claim 8, wherein thecomposition is preadsorbed onto a ceramic vehicle that is precoated withfibronectin and is suitable for implantation to augment bone marrowtransplantation.
 10. The composition as in claim 8, wherein thecomposition is suitable for use in augmenting bone marrowtransplantation.
 11. The composition as in claim 8, wherein thecomposition also comprises haemopoietic cells.
 12. The composition as inclaim 8, wherein said STRO-1^(bright) cells in the enriched populationcomprise an exogenous nucleic acid that expresses a therapeutic agenttransformed into them such that the composition may be introduced intothe body of a patient to release the therapeutic agent.
 13. The enrichedpopulation of cells as in claim 1, wherein the STRO-1^(bright) cells arenegative for at least one marker selected from the group consisting ofCBFA-1, collagen type II, PPARγ2, and glycophorin A.